Isothermal Titration Calorimetric Analysis of Membrane Protein-Protein Interactions; Cytochrome b6f - Ferredoxin Nadp+ Reductase

Abstract

Ferredoxin-NADP+ reductase (FNR) was inferred from studies with the cytochrome b6f complex isolated from spinach thylakoid and cyanobacterial membranes to be an integral subunit of the b6f complex. This inference has been examined through analysis of the interaction thermodynamics between FNR and the b6f complex. Isothermal titration calorimetry (ITC) was used to characterize the interaction of spinach FNR with the b6f complex derived from two plant sources, spinach & Zea maize. ITC did not detect interactions of significant amplitude between FNR and the b6f complex in detergent solution. The weak interaction (Kd, 90 ± 40 µM; ΔHo, −14 ± 8 kcal/mol;, ΔSo, 42 ± 18 cal/mol-oK) reported previously (Biophys Soc. Mtg, 2020) is explained by interaction of FNR with UDM detergent micelles. These results indicate a general difficulty in measurement of the energetics associated with interaction of a water-soluble protein with a membrane protein in detergent solution. Concerning the effect of detergent on the structure of water-soluble FNR, far- and near UV circular dichroism (CD), employed to determine the effect of detergent on FNR, did not reveal changes in secondary or tertiary structure. Thermal melting analysis, however, detected some loss of interaction between the two FNR domains in the presence of UDM. It is proposed that UDM micelles block the inter-domain interaction of FNR important for membrane-binding interactions, e. g., contact with the b6f complex. Support: (i) Purdue, DOE DE-SC00118238, Div. Chemical Sciences-Geosciences/Biosciences, Office of Basic Energy Sciences-U. S. Dept. of Energy; (ii) Osaka, JST-CREST (JPMJCR13M4).